Exocytosis is a fundamental cell biological process in all eukaryotic cells, through which intracellular molecules are secreted and the levels of membrane proteins and lipids are regulated. The work proposed here focuses on the exocyst, a multimeric, ~800kDa protein complex that functions in the last steps of polarized exocytosis. It helps to mediate recognition between secretory vesicles and the plasma membrane and may assist the SNARE complex in promoting membrane fusion. It is important for the insulin stimulated trafficking of the Glut4 transporter and, as an effector of the RalA GTPase, it is relevant to tumorigenesis. Yet little is known of exocyst architecture or of the mechanisms by which its assembly is tightly controlled by small GTPases and their regulators. Here we will use biophysical and biochemical approaches in exploring key interactions that promote exocyst assembly and the overall architecture of the exocyst complex. Our first aim is to explore the mechanism by which the rab GTPase Sec4p, a regulator of exocyst assembly, is activated by its guanine exchange factor (GEF) Sec2p. Once activated, Sec4p recruits the exocyst component Sec15p to the vesicle membrane. Good quality, selenomethionine substituted crystals of a complex between Sec4p and the GEF domain of Sec2p have been obtained. The mechanistic insights obtained from the Sec4p/Sec2p complex will be among the first for the activation of rab GTPases in the large rab family by GEFS. Our second aim is to study the interactions of exocyst subunits with regulators of exocyst assembly. We will investigate the interactions of the small GTPase Rho3p with its effector, the exocyst component Exo70p, and the interactions between the exocyst subunit Sec15p, an effector of the Sec4p GTPase, and Sec2p, the GEF for Sec4p. This second interaction is believed to ensure that the GEF is maintained at sites of exocyst assembly. Finally, in our third aim, we will study the overall architecture of the exocyst. Exocyst subcomplexes will be analyzed by both cryoEM and by crystallographic methods. A detailed picture of how the various exocyst subunits interact will help to formulate an understanding of exocyst assembly and function. [unreadable] [unreadable] [unreadable]